Edible plant-derived nanovesicles have been explored as effective materials for preventing colorectal cancer (CRC) incidence, dependent on gene status, as a K-Ras-activating mutation via the macropinocytosis pathway. Approximately 70% of CRC harbors the p53 mutation, which is strongly associated with a poor prognosis for CRC. However, it has not been revealed whether p53 inactivation activates the macropinocytosis pathway or not. In this study, we investigated parental cells, wild-type or null for p53 treated with Citrus limon L.-derived nanovesicles, as potential materials for CRC prevention. Using ultracentrifugation, we obtained C. limon L.-derived nanovesicles, the diameters of which were approximately 100 nm, similar to that of the exosomes derived from mammalian cells. C. limon L.-derived nanovesicles showed inhibitory effects on cell growth in not p53-wild, but also in p53-inactivated CRC cells. Furthermore, we revealed that the macropinocytosis pathway is activated by p53 inactivation and C. limon L.-derived nanovesicles were up taken via the macropinocytosis pathway. Notably, although C. limon L.-derived nanovesicles contained citrate, the inhibitory effects of citrate were not dependent on the p53 status. We thus provide a novel mechanism for the growth inhibition of C. limon L.-derived nanovesicles via macropinocytosis and expect to develop a functional food product containing them for preventing p53-inactivation CRC incidence.

Most mitochondrial proteins encoded in the nuclear genome are synthesized in the cytoplasm. These proteins subsequently undergo maturation through the cleavage of a signal sequence at the N-terminus by one or two mitochondrial signal peptidases, which is essential for their function within mitochondria. The present study demonstrates that adipocyte-specific knockout of one mitochondrial signal peptidase, mitochondrial intermediate peptidase (MIPEP), resulted in disordered mitochondrial proteostasis of MIPEP substrate proteins and their defective maturation. MIPEP deficiency in white and brown adipocytes suppressed the expression of adipocyte differentiation, lipid metabolism, and mitochondrial biogenesis genes. These alterations led to lipoatrophy in white adipose tissue and the whitening of brown adipose tissue. Additionally, it induced an atypical mitochondrial unfolded protein response and local inflammation in white and brown adipose tissue. Furthermore, it induced fatty liver and splenomegaly and caused systemic impairments in glucose metabolism and inflammation. These findings indicate that maturation defects of certain mitochondrial proteins and subsequent proteostasis disorders in white and brown adipocytes cause chronic and systemic inflammatory and metabolic dysfunctions.

Peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1α) is a master transcriptional cofactor for mitochondrial biogenesis. Mitochondrial intermediate peptidase (MIPEP), a mitochondrial signal peptidase, plays an important role in the maturation and activation of mitochondrial proteins. Caloric restriction has lifespan‐extending effects that are reportedly exerted through induced expression of PGC‐1α and MIPEP in white adipose tissue. To evaluate how upregulation of PGC‐1α and MIPEP contributes to changes in the cellular characteristics of adipocytes, this study examined the mitochondrial function and differentiation of 3T3‐L1 preadipocytes with single overexpression (OE) or double OE of Pgc‐1α and Mipep. Compared with single‐OE cells, double‐OE cells exhibited no significant changes in oxygen consumption rate or mitochondrial morphology, but did show increased mitochondrial DNA levels. White adipocyte cell differentiation was suppressed in both Pgc‐1α single‐OE cells and double‐OE cells. Notably, double‐OE cells exhibited increased mRNA levels of phosphoethanolamine/phosphocholine phosphatase 1 (Phospho1), which plays a role in phospholipid metabolism and non‐canonical thermogenesis. Phospho1 expression was also increased in white adipose tissue of mice under caloric restriction. In summary, the double OE of Pgc‐1α and Mipep induced Phospho1 expression and suppressed adipocyte maturation, with little effect on mitochondrial function. This study provides new insights into the mitochondria‐related mechanism of caloric restriction in adipocytes.

Mitochondrial intermediate peptidase (MIPEP) is a mitochondrial signal peptidase that removes N-terminal amino acids from mitochondrial matrix proteins. We have identified a novel Mipep splice variant that lacks exons 15 and 16, which we termed “ΔMIPEP”. We characterized the molecular features of ΔMIPEP by investigating its expression level in numerous mouse tissues and by performing a computer simulation that allows the prediction of protein structures and substrate-binding properties. ΔMipep mRNA was detected in all mouse tissues examined but at much lower levels than full-length Mipep. Structure prediction and docking simulation of full-length MIPEP and ΔMIPEP with substrates of MIPEP, such as malate dehydrogenase 2 (MDH2) and cytochrome c oxidase subunit 4, showed that entry of these substrates into ΔMIPEP with a low binding energy was greatly restricted. To determine levels of MIPEP substrates in the presence or absence of full-length MIPEP or ΔMIPEP, we created Mipep and ΔMipep overexpression 3T3-L1 cells and Mipep knockout (KO) cells. Western blotting showed that in Mipep KO cells Mipep overexpression slightly decreased the molecular weight of MDH2 and Sirtuin 3, another MIPEP substrate, whereas ΔMipep overexpression did not. These results indicate that ΔMIPEP fails to recognize MIPEP substrate proteins. Together, our findings indicate that ΔMIPEP is a novel splice variant that can contribute to mitochondrial signal peptidase-mediated regulation of mitochondrial protein homeostasis.